Probing phospholipid transport by the Pqi pathway

The PqiABC complex has been implicated in phospholipid (PL) transport between the inner and outer membranes of gram-negative bacteria. This one complex spans the periplasm with components in both membranes, tethering them together. In a recent experiment, RB2220703, we proposed utilising this trans-envelope complex as a scaffold to create a planar double membrane architecture, providing a unique gram-negative envelope mimetic to probe envelope biology. Our Preliminary Neutron Reflectometry (NR) experiments confirmed its successful creation. Furthermore, we predicted PqiABC to be a proton motive force (PMF) driven PL pump based on structural homology. By altering the buffer pH, we saw tantalising evidence that we were able to emulate the PMF leading to a time dependent movement of phospholipids from the outer to the inner membranes, consistent with PMF driven PL transport. However repeat experiments (RB2320187) failed to repeat our earlier observations, we have since identified complex dissociation as the likely cause. In this proposal, we wish to study system formation and activity of a functionally active fused construct that prevents system dissociation with the hope this will improve activity and stability of the system. If proven it will be the first direct evidence of phospholipid transport from the inner to the outer membrane in gram-negative bacteria.